Mechanisms of mutagenesis and maintenance of genetic information in mammalian cells

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It is important for organisms to keep their genetic information intact through generations, and this is accomplished by accurate DNA replication and efficient repair of damaged DNA.We investigated the molecular mechanisms underlying these processes.1. Molecular cloning of the genes encoding the proteins that suppress the occurrence of induced mutations and elucidation of the role of the protein : We have cloned the cDNAs of human, mouse, rat and rabbit O^6-methylguanine-DNA methyltransferases, which play a central role in repair of DNA methylated by alkylating agents and in preventing the occurrence of mutations and formation of tumors, and determined their nucleotide sequences. We found the conserved amino acid sequences around the active sites of the enzymes by introducing the amino acid substitutions. We have detected only a very low level of expression of the gene in certain cell lines, which are often established from tumors. There must be an abnormality elsewhere in some steps of … More gene expression since we could demonstrate that initiation of transcription of the gene occurs at the same level in the defective cells as in the normal cells. We have cloned the mouse genomic sequences and constructed targeting vectors for elucidation of role of the enzyme in tumorigenesis by making defective mice.2. Mechanisms of keeping high fidelity of DNA replication : To understand the controlling mechanisms for spontaneous mutagenesis we require the complete knowledge of the molecular mechanisms of attaining the high fidelity of DNA replication. We focused our studies to proteins found in bacteria to be responsible for keeping the mutation frequency at very low level. We found that human cell extracts contain an activity of 8-oxo-dGTPase similar to E.coli MutT protein. We purified the protein and determined the partial amino acid sequences of the protein and cloned human cDNA by RT-PCR.Cloning and analysis of the cDNA and genomic sequences of relevant organisms were carried out. Less